Plaque inhibiting oligosaccharide

ABSTRACT

A purified oligosaccharide consisting of a hexasaccharide with a native molecular weight of 959 which can be isolated from the cell walls of Streptococcus oralis. The oligosaccharide is able to significantly block the interaction between the human oral plaque bacteria Streptococcus oralis H1 and Capnocytophaga ochracea ATCC 33596. This purified oligosaccharide contains saccharide components found to inhibit many know interactions between plaque bacteria and may be effective in prevention, inhibition and reversal of dental plaque deposits. The oligosaccharide may be applied effectively when incorporated in toothpastes, mouth wash, etc.

This application is a divisional of application Ser. No. 349,772, filedon May 10, 1989, now U.S. Pat. No. 5,071,977, the entire contents ofwhich are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a purified oligosaccharide which can beisolated from a natural source, e.g. from the cell wall polysaccharideof Streotococcus oralis. S. oralis is found in significant numbers inhuman dental plaque. This oligosaccharide is located at the site where adifferent oral plaque bacteria attaches, and contains saccharidecomponents which are effective in blocking attachment of many plaquebacteria to each other. Use of this oligosaccharide may reverse theattachment of many plaque bacteria in the human oral cavity. For thisuse, the oligosaccharide is incorporated in tooth paste, mouthwash, etc.The oligosaccharide is a hexasaccharide with a molecular weight of 959.

BACKGROUND OF THE INVENTION

Most bacteria isolated from the human oral cavity possess the ability toparticipate in intergeneric coaggregation (bacteria from differentgenera bind to each other primarily via a protein on one attaching to asaccharide component on the other). Coaggregation is characterized by ahighly specific binding between stable surface components found on twodifferent bacterial types. Intergeneric coaggregation is thought to playan important role in the formation of dental plaque deposits (10, 11).Streptococcus oralis is one of the earliest colonizers of the cleantooth surface and is found in significant numbers in dental plaque (4,19). The interaction between Streptococcus oralis H1 ATCC55229 andCapnocytophaga ochracea ATCC 33596 was first described by Kolenbranderand Andersen (9). A study (22) demonstrated that L-rhamnose and D-fucosewere the most effective inhibitors of this coaggregation whilegalactosides, i.e. B-methyl galactoside, D-galactose, lactose ando-methyl galactoside were less effective inhibitors.

Thus far, the only bacterial carbohydrate receptor for a bacteriallectin studied in detail is the carbohydrate receptor on Streptococcusoralis 34 that is recognized by the adhesin on Actinomyces viscosusT14V. This interaction is inhibited by β-galactosides andB-N-acetylgalactosaminides. A cell wall coaggregation-inhibitingpolysaccharide antigen from Streptococcus oralis 34 has been isolatedand characterized (16). This polysaccharide consists of rhamnose,glucose, galactose and N-acetyl galactosamine in a hexasacchariderepeating unit (15). The polysaccharide inhibits coaggregation, containssaccharide components that by themselves are effective inhibitors of theinteraction, and is a major cell surface antigen (16).

The available technology in combating dental plaque comprises the use oftoothpastes, mouth washes, chewing gum, etc., which all work in anon-specific fashion, primarily by means of detergents and abrasives.

DETAILED DESCRIPTION OF THE INVENTION

The present invention works at the molecular level at the exactattachment site of two bacteria.

The present invention is based on the fact that an isolated and purifiedcomponent of the cell wall polysaccharide from Streotococcus oralis H1serves as a receptor for the adhesin on Capnocytophaga ochracea ATCC33596. Criteria similar to those utilized in the studies of McIntire etal., (14, 16), i.e., the isolation of a polysaccharide antigen and itsconstituent oligosaccharide, which both inhibit coaggregation andcontain monosaccharide components which serve as effective inhibitors ofcoaggregation, were applied to the identification and characterizationof the Streptococcus oralis H1 carbohydrate receptor. The absence ofthis coaggregation-inhibiting polymer and its hexasaccharide repeatingunit in a coaggregation-defective mutant of Streotococcus oralis H1supported the conclusion that the polysaccharide isolated from the wildtype strain was indeed the carbohydrate receptor.

An object of the present invention is to provide an noveloligosaccharide which can be isolated from the cell walls ofStreptococcus oralis H1, said oligosaccharide consisting of ahexasaccharide of the structural formula I: ##STR1##

A further object of the invention is to provide a process for thepreparation of the above-defined hexasaccharide, said process comprisesthe steps of a) isolating crude cell walls of Streptococcus oralis H1cells, b) liberating cell wall polysaccharide, c) optionally purifyingthe polysaccharide, d) hydrolysing the polysaccharide, e) isolating thedesired product.

A further object of the invention is to provide a method of preventing,inhibiting or reversing the build-up of human dental plaque deposits byadministering a composition comprising the hexasaccharide, a compositionfor preventing, inhibiting or reversing the build-up of human dentalplaque deposits comprising the hexasaccharide, a method of inhibitingcoaggregation between Streotococcus oralis H1 and Capnocytophagaochracea, especially of the strain ATCC 33596, by preincubation of thehexasaccharide with the C. ochracea partner and finally a method forblocking the adhesin from the gram-negative bacteria Capnocytophagaochracea by incubating the bacteria with the hexasaccharide.

As the oligosaccharide of the present invention appears to haveexcellent dental plaque prophylactic activities, it can be applied tohuman teeth for the purpose of the prevention of dental plaque byconventional methods, conventional types of unit dosages or withconventional carriers or diluents. A preferred composition is a toothpaste or a mouth wash. The conventional carriers or diluents are, forexample, water, tooth powder, toothpaste, chewing gum, ointment, and thelike. The hexasaccharide of the present invention is incorporated intothe compositions in an amount of 0.001 to 5% by weight, preferably 0.05to 1% by weight.

In the preparation of toothpaste and tooth powder containing the presenthexasaccharide, conventional vehicles are used unless they giveessentially undesirable effects to the activity of the hexasaccharide.An appropriate water-insoluble polishing agent can be incorporated inthe toothpaste and tooth powder. Suitable examples of the polishingagents are dicalcium phosphate, tricalcium phosphate, and the like.These polishing agents usually constitute a major proportion by weightof the solid ingredients. The content of the polishing agents ispreferably about 30 to 60% by weight of the total composition intoothpaste and 85 to 95% by weight in tooth powder.

In the preparation of toothpaste, some plasticizers may also be added.Suitable examples of plasticizers are water, glycerin, sorbitol,propylene glycol, monoglyceryl stearate, white petroleum jelly, cetylalcohol, and the like, or a mixture thereof. The toothpaste ispreferably formulated with a gelling agent, such as sodium carboxymethylcellulose, hydroxyethyl cellulose, polyvinyl pyrrolidone, gumtragacanth, and the like, and may also optionally be formulated withother additional components, such as flavors, sweetening agents andcoloring agents.

In the preparation of chewing gum containing the present hexasaccharide,conventional gum base such as chicle resin, polyvinyl acetate, and thelike may be used. The chewing gum may also be formulated with otherconventional vehicles, such as plasticizers, softeners, sweeteningagents, flavors and coloring agents.

Other means for using the present hexasaccharide is a form of ointment.To the teeth to be treated an ointment containing the presenthexasaccharide is applied, whereafter the teeth are being rubbed by afinger or a toothbrush. The ointment can be prepared by conventionalmethod using conventional vehicles which can be applied to the mouthunless they show inhibitory or destructive action onto the presenthexasaccharide. Suitable examples of materials to be used as an ointmentbase are sodium carboxymethyl cellulose, hydroxyethyl cellulose andPlastibase 50 W (dental paste base made by Squibb Co., Ltd.) which canform a jelly-like or creamy ointment.

The present hexasaccharide can also be used in the form of a chewabletablet or troche. When the chewable tablet or troche containing thepresent hexasaccharide is kept in the mouth, the hexasaccharide will bein contact with teeth for a long period of time. The chewable tablet ortroche can be prepared by conventional methods using conventionalvehicles, such as mannitol and sorbitol, and other conventionallubricating agents, sweetening agents, coloring agents, and the like.

The present hexasaccharide may also be admixed with confectionary suchas candy, cake, and the like.

The hexasaccharide which blocks the coaggregation between Streptococcusoralis H1 and Capnocytophaga ochracea can be released from the walls ofthe gram-positive streptococcus cells by means of two methods,autoclaving and mutanolysin treatment, the latter has proved to besignificantly more effective, ultimately yielding 2 to 3 fold higherquantities of purified material. For convenience, the two methods aredesignated below as AC (autoclaving) and ML (mutanolysin); the latterhas two variants, I and II, so that the two mutanolysin-based variantsare designated ML-I and ML-II, respectively. The methods are explainedin detail in the section "Materials and Methods". The polysaccharidereleased from the streptococcal cell walls by either treatment yield theoligomer, which is present as repeating units of hexasaccharide,consisting of three hexoses, rhamnose, galactose and glucose, in theratio of 2:3:1, respectively. The hexasaccharide units in thepolysaccharide appear to be linked to one another by phosphodiesterbonds. The purified hexasaccharide is a four fold more potent inhibitorof coaggregation than the native polysaccharide. Studies with acoaggregation-defective mutant of Streptococcus oralis H1 revealed thatthe cell walls of the mutant contained neither the polysaccharide northe hexasaccharide repeating unit. The purification of a polysaccharideand its hexasaccharide repeating unit which both inhibited coaggregationand the absence of this polysaccharide or hexasaccharide on acoaggregation-defective mutant demonstrate that the hexasaccharidederived from the polysaccharide functions as the receptor for theadhesin from Capnocytophaga ochracea ATCC 33596.

MATERIALS AND METHODS

All cells from the bacteria Streotococcus oralis H1 and Capnocytophagaochracea ATCC 33596 were grown under anaerobic conditions. Streotococcusoralis H1 was grown in a complex medium containing tryptone, yeastextract, Tween 80 and K₂ HPO₄ with 0.3% glucose (17), and Capnocytophagaochracea ATCC 33596 was cultivated in Schaedler broth (BBL, MicrobiologySystems). A coaggregation-defective mutant of Streotococcus oralis H1,Streotococcus oralis PK1831, was provided by Dr. E. Weiss (Tel AvivUniversity, Israel).

Coaggregation-inhibition assays. Coaggregation assays with intact cellsof Streotococcus oralis H1 and Capnocytophaga ochracea ATCC 33596 wereconducted as described in (5). Initial screening forcoaggregation-inhibiting material removed from Streptococcus oralis H1cells was done using a semiquantitive microtiter plate assay. Twenty μ1of two fold serial dilutions of the potential inhibitor in coaggregationbuffer (1 mM Tris, 0.1 mM CaCl₁, 0.1 mM MgCl₂, 0.15 M NaCl, 0.02% NaN₃,pH 8.0) was placed into 96 well microtiter plates (Falcon, B-D, Oxnard,CA) and equal volumes of Capnocytophaga ochracea ATCC 33596 (at a celldensity of about 1×10⁹ cells/ml, 260 Klett units, red filter,Klett-Summerson, New York, NY) were added. After gentle vortex-mixing,the plates were incubated at room temperature (45 min). An equal volumeof Streptococcus oralis H1 at the same cell density was added, gentlyvortex-mixed, incubated for 30 min and visually scored for the degree ofcoaggregation with the aid of a microtiter plate illuminator (Dynatech,Alexandria, VA). The half maximal inhibitory concentration was definedas that amount of inhibitor required to reduce coaggregation by 50% ofthe control value (buffer substituted for inhibitor). Quantitativeinhibition assays were carried out according to the procedure describedin Weiss et al. (22). To verify that the isolatedcoaggregation-inhibiting polysaccharide (CIP) was responsible for theobserved inhibition, the purified polysaccharide was oxidized with l0mMsodium periodate at a pH of 4 for 1 hr at room temperature (13) andtested for its ability to inhibit coaggregation in the quantitativeinhibition assay.

Chemical assays. Total hexose was assayed by the phenol-sulfuric acidmethod (7), inorganic and total phosphate by the method of Ames (1),protein was assayed by a dye-binding method (2), and nitrogendetermination according to the method of Schiffman et al. (18).

Polysaccharide release from S. sanquis H1 cell walls. Polysaccharide wasremoved from S. oralis H1 cell walls in principle by two differentmethods as explained above, in the autoclaving (AC) and the mutanolysinmethods (ML) of which the mutanolysin method has two variants, ML-I andML-II. In both the AC and the ML-I methods the polysaccharide ispurified, then the hexasaccharide is isolated from the hydrolysisproducts after the purified polysaccharide is degraded with hydrofluoricacid (HF). The ML-II method is similar to the ML-I method, but thepurification of the polysaccharide is bypassed with the goal ofobtaining only the hexasaccharide in high yield. The entire purificationprocess can be summarized as having three stages:

Stage 1: Removal of the polysaccharide from the S. oralis cell wall

This is (in the AC method) effected by autoclaving (which is known tobreak the chemical bond between the bacterial cell wall and some cellwall polysaccharides, solubilizing the polysaccharides) and (in the ML-Iand ML-II methods) by mutanolysin (an enzyme that cleaves the cell wallinto small pieces, with the cell wall polysaccharide solubilized by theenzymatic action). The two mutanolysin methods, ML-I and ML-II,respectively, are identical in Stage 1. In Stage 1 the cell walls (inthe AC method) or the whole cells (in both of the ML-methods) are washedwith a detergent and an enzyme solution. As a detergent Triton can beused, as an enzyme Pronase can be used, the latter in a concentration of0.05-1%, especially about 0.1-0.2%. In the ML methods the cells areadvantageously washed with a strong salt solution in order to denatureand remove adherent protein and lipid; an especially preferred saltsolution is a quanidine hydrochloride solution in a strength of from 3to 7 M, preferably at least 4 M, especially 6 M.

Stage 2: Purification of the polysaccharide from other components of theautoclaving- or mutanolysin-derived extract

The purification of the polysaccharide from the AC or ML-I methodsderived extract was accomplished by anion exchange chromatography (on aMono Q column). Purity was demonstrated by immunoelectrophoresis of thepolysaccharide with development of the electrophoresis gel with antibodyto whole S. oralis H1 cells. The polysaccharide purification proceduresfor cell wall derived material extracted by AC and ML-I methods wereidentical. Stage 2 was bypassed in the ML-II method.

Stage 3: Purification of the hexasaccharide from the polysaccharidehydrolysate

In all three methods the hexasaccharide was purified by HPLC on an aminobonded silica column (NH₂ silica), and the purity demonstrated by HPTLC.The AC and ML-I methods in Stage 3 were identical. The ML-II method wasdifferent in that the mutanolysin extract from Stage 1 was treated withHF and the hexasaccharide purified from the hydrolysate of the totalextract (instead of the hydrolysate of the polysaccharide).

The benefits of the ML-I method in relation to the AC method are a highyield of purified polysaccharide and subsequent hexasaccharide ifdesired, and this procedure provides a simple, effective way to scale upin order to isolate greater quantities of the purified materials.

The ML-II procedure provides a simple, highly efficient way to isolatepurified hexasaccharide in milligram quantities with the least amount ofeffort of the three methods. This variation on the mutanolysin method isunique and constitutes a preferred variant of the process according tothe invention.

The autoclaving method was based on the hydrolysis of the phosphodiesterlinkage between polysaccharide and peptidoglycan by exposure to hightemperatures (3) and was a variation of that reported by McIntire et al.(16). Washed whole cells suspended in 25 mM Tris-HCl buffer, pH 8.0 wereultrasonically disrupted (Branson, 2 cycles of 6 min each, constantcooling) and the cell walls were obtained from the layer above theintact cell pellet following centrifugation (25,000×g for 20 min). Afterwashing, the isolated cell walls were treated with 0.1% Triton-X 100(membrane purity grade, Boehringer Mannheim), in the Tris HCl buffer(16h, 4° C.), washed thoroughly in buffer without detergent, incubatedin 0.2% Pronase (Calbiochem, La Jolla) dissolved in the Tris HCl buffer(two cycles of 2.5h, at 37° C.), and washed. The treated cell walls werethen autoclaved in deionized water for 60 min at 6.8 kg/cm² pressure.

The second method, treatment with mutanolysin, (ML-I), has been employedsuccessfully with S. oralis 34 (6). Intact S. oralis H1 cells weresequentially treated with 0.1% Triton X-100 in Tris HCl buffer (72 h, at4° C.); 0.1% Pronase (Calbiochem) in Tris HCl buffer (two cycles of 2.5h each at 37° C.); and 6M guanidine HCl for 48-72 h. at 4° C., withextensive washing after each treatment. This crude cell wall preparationwas then digested by incubation with 26 μg/ml (corresponding to 4 mg/ginitial wet weight S. oralis H1 cells) mutanolysin.

Cell wall material of the coaggregation-defective mutant were obtainedby the mutanolysin method (equivalent to ML-I).

Isolation of coaggregation-inhibiting material. Material released fromisolated S. oralis H1 cell walls by the autoclaving method was clarifiedby centrifugation at 25,000×g for 20 min followed by ultracentrifugationat 300,000×g, for 90 min. More non-CIP material was removed byprecipitation by lowering the pH to 1.5-2.0 with 4M HCI (16). The pH ofthe polysaccharide solution was readjusted to 6.0-6.5 with 4M NaOH,dialyzed against deionized water and lyophilized.

Polysaccharide released from crude cell walls by the mutanolysin methodML-I was clarified by centrifugation at 25,000 ×g for 20 min and thesupernatant was adjusted to a concentration of 5% trichloroacetic acid(TCA) by the addition of 50% TCA. After centrifugation at 25,00033g for20 min., the supernatant was neutralized by adding solid Tris, dialyzedextensively against water and lyophilized.

The autoclaving extract and the mutanolysin (ML-I and ML-II) extractwere both assayed for their ability to inhibit coaggregation in asemiquantitive microtiter plate assay.

Hydrofluoric acid treatment. Solutions containing 10 mg/ml of crudepolysaccharide preparation (from ML-II), purified polysaccharide (fromAC and ML-I) or purified oligosaccharide (from AC, ML-I and ML-II) weretreated with 48% hydrofluoric acid (HF, Baker) for 4 days at 4° C (23).HF was removed by 4 or 5 cycles of lyophilization or evaporation(Speed-Vac, Savant) followed by rehydration with distilled, deionizedwater. The rate and efficacy of hydrolysis was monitored by treatingaliquots of purified polysaccharide with 48% HF for 30 min, 1 h, 4 h, 8h, 24 h, 48 h, 72 h, 96 h, and 144 h in a time course experiment; HF wasremoved as above.

Isolation of S. oralis H1 CIP and HF released oligosaccharide.Coaggregation-inhibiting material released from S. sanguis H1 cell wallsby both the autoclaving and the mutanolysin method ML-I was furtherpurified by anion exchange chromatography on a Mono Q HR 5/5 FPLC column(Pharmacia) mounted on a Hewlett-Packard 1090L high performance liquidchromatograph (HPLC). Polysaccharide was eluted at a flow rate of 0.5ml/min with a 0.1 to 0.25 M NaCl gradient prepared in 2 mM Tris-HCl, pH8.0. All fractions were monitored for presence of neutral hexose by thephenolsulfuric acid assay (7).

The oligosaccharides released by HF treatment of polysaccharide purifiedon the Mono Q column were separated from partially digested startingmaterial by molecular sieve chromatography on a 2.4×90cm P2 column(BioRad) using distilled water as the eluent; the flow rate was 25 ml/h.Fractions from the P2 column were pooled, lyophilized, resuspended in amixture (75%: 35%) of acetonitrile (ACN, Burdick and Jackson) and water(HPLC grade, Burdick and Jackson) and applied to 8 mm x 30 cm MicroPakAX-5 (Varian) diaminopropyl bonded silica HPLC column (24). The majoroligosaccharide was eluted by passing a linear gradient of water throughthe column. The gradient was initiated after washing the column for 15min with the initial ACN:water mixture; the gradient was formed byincreasing the aqueous phase with 1.75% water/min. at a flow rate of 1ml/min.

Gas-liquid chromatography. Oligosaccharide purified by HPLC washydrolyzed with 4M trifluoroacetic acid (TFA) and the monosaccharidesconverted to their respective alditol acetate derivatives (8). Standardswere purchased as alditol acetate derivatives (Biocarb, Lund, Sweden) orderivatized from monosaccharides (8). Samples and standards wereanalyzed by gasliquid chromatography (Hewlett-Packard, model 5840A)fitted with a capillary inlet system and flame ionization detector. A0.25 mm ×15 cm SP2330 cyanopropyl bonded phase capillary column(Supelco) was used to resolve the alditol acetate derivatives of themonosaccharides. The injector and detector temperatures were held at300° C. and the oven was maintained at a constant temperature of 220° C.The carrier gas, helium, was flushed through the column at a flow rateof approximately 2.5 ml/min.

High performance thin-layer chromatography. All samples including theCIP, HF released oligosaccharides, and monosaccharides derived from theHPLC purified oligosaccharide were applied to Silica 60 high performancethin-layer chromatography (HPTLC) 10×10 cm glass backed plates (EMScience, Cherry Hill, N.J.), and resolved using a mobile phaseconsisting of chloroform, methanol and water in a ratio of 10:10:3 (byvolume) (21). Constituents in the samples were visualized by sprayingthe plates with a freshly prepared solution of naphthoresorcenol (20mg), sulfuric acid (0.2 ml) and ethanol (10 ml) (12).

Immunoassays. Immunoelectrophoresis was carried out according to themethod of Wang (20). A solution containing 1% agarose (electrophoresisgrade, BRL) in barbital buffer was poured onto gelbond (FMC, Rockland,Maine) support medium and 15 μl samples containing roughly 5 μg of theappropriate preparation were loaded into wells and were subjected toelectrophoresis. Troughs were filled with 150 μl antisera raised towhole, intact S. sanguis HI cells and allowed to develop. Gels werestained in 0.1% amido black.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and 1B. Elution profile and immunoelectrophoresis of selectedfractions of polysaccharide (mutanolysin releasedcoaggregation-inhibiting material) eluted from HPLC anion exchangechromatography. A) Lyophylized coaggregation-inhibiting material wasresuspended in 2 mM Tris, 0.1M NaCl and applied onto a Mono Q anionexchange column. Coaggregation-inhibiting material was eluted with asalt gradient of 0.1-0.25M NaCl ( -μg hexose/fraction; □- mM NaCl). B).Immunoelectrophoresis stained with 0.1% amido black. Numbers refer tofraction numbers of A). Troughs were filled with rabbit antisera towhole S. oralis H1 cells.

FIG. 2. Isolation of hydrofluoric acid released major oligosaccharide.P2 pooled, HF released material was applied to a MicroPac AX-5 HPLCcolumn and eluted with a gradient of increasing water (□-μghexose/fraction; % H₂ O). See Materials and Methods for chromatographyconditions.

FIG. 3. HPTLC of CIP (B); HF treated CIP (C); HPLC purifiedoligosaccharide (D); and HPLC purified oligosaccharide hydrolyzed tomonosaccharides (E) by trifluoroacetic acid (4M, 4h, 100° C) run onSilica G HPTLC. See Materials and Methods for chromatography conditions.Standards A) Equimolar mixture of rhamnose, glucose and galactose(fastest (top) to slowest migrating, respectively); F) maltopentaose; G)maltohexaose. 16 μg sample applied to lane C); all other 2-8 μg/lane.

FIG. 4. HPTLC of HF treated CIP in a time course experiment, and thepurified oligosaccharide after HF treatment. (A) CIP; HF treated CIP intime course experiment (B-K; 0.5, 1, 2, 4, 8, 24, 48, 72, 96, and 144hours, respectively). (L) HPLC purified oligosaccharide (M) HPLCpurified oligosaccharide after HF treatment (4d, 4° C); N) Equimolarmixture of rhamnose, glucose and galactose. Arrow indicates position ofthe major oligosaccharide component.

FIGURE 5. HPTLC of mutanolysin-released, HF treated (4d, 4° C.),polysaccharide material from coaggregation-deficient mutant S. oralisPK1831 and wild type S. oralis H1. B) Wild type mutanolysin-releasedmaterial and HF treatment; C) Mutant mutanolysin-released material afterHF treatment. Standards A) Equimolar mixture of rhamnose, glucose andgalactose; D) HPLC purified oligosaccharide of wild type origin.

FIG. 6. Quantitative inhibition of coaggregation between S. oralis H1and C. ochracea ATCC 33596 with S. oralis H1 cell wall mutanolysinextract (◯), purified CIP (□), and purified oligosaccharide (Δ). 50%inhibition concentrations for the mutanolysin-released material, thepurified polysaccharide and the purified oligosaccharide were 5.10, 1.79and 0.44 mg/ml respectively.

RESULTS

Purification of coagregation-inhibiting polysaccharide (CIP). Thematerials released by mutanolysin treatment and by autoclaving weretested in a semiquantitative coaggregation-inhibition assay for theirability to prevent the interaction of the two partner cells and wereanalyzed for their chemical content. Half-maximal inhibition ofcoaggregation between the two partner cells took place at 4.0 mg/ml and4.4 mg/ml for the mutanolysin extract and the autoclaving extract,respectively. The results are shown in Table I.

                  TABLE 1                                                         ______________________________________                                        INHIBITION ACTIVITY AND CHEMICAL ANALYSES                                     OF AUTOCLAVING AND MUTANOLYSIN EXTRACTS                                       AND ION EXCHANGE PURIFIED POLYSACCHARIDE                                      RELEASED FROM Streptococcus oralis H1 CELL WALLS                                        Inhibi-                                                                       tion.sup.1                                                                           Hexose.sup.2                                                                           Phosphate Nitrogen                                            (mg/ml)                                                                              (wt %)   (wt %)    (wt %)                                    ______________________________________                                        A.                                                                            Mutanolysin Extract                                                                       4.0      81.6     6.3     2.5                                     Peak 2a.sup.3                                                                             1.7      86.7     8.8     0.7                                     Peak 2b     1.7      85.3     9.0     0.7                                     B.                                                                            Autoclaving Extract                                                                       4.4      84.5     7.1     1.9                                     Peak 2a.sup.4                                                                             1.7      82.6     8.6     0.5                                     Peak 2b     1.7      80.1     7.4     0.9                                     ______________________________________                                         .sup.1 Value range reported as approximate mg amount necessary to give        half maximal inhibition in microtiter inhibition assay.                       .sup.2 Chemical assays as described in Materials and Methods. Phosphate       was present as organic phosphate. Protein assays indicated that protein       levels were below the level of detection in all samples (less than 0.5%).     .sup.3 Peak 2a refers to fractions 15-20 and Peak 2b refers to fractions      21-27 in FIG. 1A.                                                             .sup.4 Peak 2a refers to fractions 16-21 and Peak 2b refers to fractions      22-29 from a column run under conditions identical to those of FIG. 1A.  

When compared by chemical assays, only slight differences were foundbetween the two preparations released from S. sanquis H1 cell walls. Thematerials consisted primarily of neutral hexose 81.6-84.5% by weight ofinitial lyophylized material), while organic phosphate was present inthe range of 6.3 to 7.1%. The content of nitrogen was low (1.9-2.5%),and protein assays indicated no detectable protein.

Polysaccharide released by either method was eluted from a Mono Q anionexchange chromatography by applying a NaCl gradient. The elutionprofiles for the mutanolysin extract (FIG. 1A) and the autoclavingextract (not shown) were very similar. In both instances,sugar-containing material was eluted with a NaCl concentration between130-200 mM. Samples were taken from selected areas across the peak,subjected to immunoelectrophoresis (FIG. 1B), and developed with anti-S.oralis H1 serum to determine whether any of the fractions contained cellsurface-associated antigens. Multiple immune precipitin arcs appeared inthe initial flow-through material (Peak 1 of FIG. 1A, fractions 1-6)while the eluted material (Peak 2, fractions 15-27) consisted of asingle antigen. While a single antigen was present in the salt-elutedpeak (Peak 2) for both the mutanolysin-released material and theautoclaving-released material (not shown), the fractions comprising thepeak were divided into two lots (forward, Peak 2a, and latter, Peak 2b,portions of the peak) and compared for inhibitory activity and chemicalcontent, Table 1. Peak 2a was essentially the same as Peak 2b by allcriteria tested, and the Mono Q purified mutanolysin extract (Peak 2aand 2b) appeared to be identical to the Mono Q purified autoclavingextract. Peak 2a and 2b polysaccharides were more effective inhibitorsthan the starting materials before ion exchange purification. Thenitrogen detected in the assays did not appear attributable to proteinand was most likely due to contaminating peptidoglycan.

CIP was released from cells irrespective of the method used, howeverlosses of cell wall material occurred at several steps during theautoclaving protocol. For example, sonication failed to remove all ofthe wall material from cells and the first centrifugation step did notrecover the smaller cell wall components. After autoclaving, largerparticles of cell wall capable of agglutinating C. ochracea ATCC 33596were removed only by ultracentrifugation. The losses that occurredduring the early steps of the autoclaving procedure were not manifestedin the mutanolysin method. The initial steps in the mutanolysinprocedure removed much of the cell's protein and lipid componentsleaving a crude cell wall preparation (probably representing thepeptidoglycan skeleton), prior to digestion by the muramidase. Recoveryof CIP after mutanolysin treatment was 2-3 fold greater than with theautoclaving method (data not shown).

Subsequent studies were conducted with polysaccharide preparationsderived by mutanolysin treatment.

Purification of HF released oligosaccharide. Anion exchange purified CIPwas HF treated and passed over a P2 column to separate unhydrolyzed andpartially hydrolyzed polysaccharide from lower molecular weight material(less than 2kD). The lower molecular weight material was pooled andapplied to a MicroPak AX-5 HPLC column. The major oligosaccharidecomponent was eluted at a ratio of approximately 60:40, water to ACN(FIG. 2). The oligosaccharide was resolved on Silica 60 HPTLC, andcompared to the intact and HF treated polysaccharide (FIG. 3, B-D). Theintact CIP (FIG. 3B) did not migrate from the origin under thesechromatographic conditions; after treatment with HF (4d, 4° C.) the CIPwas hydrolyzed to smaller components, with one prominent majoroligosaccharide component (FIG. 3C). This component was isolated fromthe other hydrolysis products by HPLC (FIG. 3D). When compared toreference oligosaccharides, maltopentaose and maltohexaose (FIG. 3F and3G, respectively), the major oligosaccharide migrated with a mobilitybetween the two standards, suggesting that this oligosaccharide waseither a pentasaccharide or hexasaccharide. When the purifiedoligosaccharide was hydrolyzed with TFA and resolved by HPTLC (FIG. 3E),three hexose components were resolved which comigrated with thereference standards rhamnose, galactose, and glucose (FIG. 3A).

Hydrolysis of Polysaccharide and purified oligosaccharide. In a timecourse experiment extending from 30 min to 6 days, hydrolysis of thepurified CIP (FIG. 4A) gave rise to one discrete oligosaccharidecomponent (denoted by arrow) and large amounts of partially hydrolyzedpolysaccharide after only 30 min incubation (FIG. 4B). With increasinglength of HF exposure, more of the non-migrating CIP was hydrolyzed tothe major oligosaccharide, and components smaller and faster migratingthan the major oligosaccharide became more prominent (FIG. 4 C-K; ascompared to monosaccharide standards, FIG. 4N). To maximize the yield ofthe major oligosaccharide (approximately 70% conversion ofpolysaccharide to oligosaccharide), a 4 day exposure of polysaccharideto HF was required (FIG. 4J). Extending the exposure to 6 days resultedin increased internal hydrolysis of the major oligosaccharide (FIG. 4K).Interestingly, the polysaccharide was resistant to hydrolysis by 2Nsulfuric acid for periods up to 8 h at a temperature of 100° C. (notshown). The relative ease with which HF released the oligomeric productfrom the purified polysaccharide as well as the resistance to hydrolysisby sulfuric acid suggested that the constituent oligosaccharide wascoupled by phosphodiester bonds. The rapidly migrating components seenon HPTLC plates (FIG. 4, lanes G-K) appear derived from the majoroligosaccharide since the HPLC purified oligosaccharide (FIG. 4L)yielded identical products when treated with HF (4d, 4° C.) (FIG. 4M).The most rapidly migrating component appears to be free rhamnose.

Composition of the oligosaccharide. Gas-liquid chromatographic analysisof the alditol acetate derivatives of the TFA hydrolyzed oligosaccharideverified the putative carbohydrate composition deduced by HPTLC. Theoligosaccharide was found to contain rhamnose, galactose and glucose inthe approximate molar ratios of 2:3:1, respectively (rhamnose 1.56-2.10:galactose 3: glucose 0.86-1.38). Analysis of the alditol acetatederivatives of the material collected as Peak 2a and 2b material (seeFIG. 1) revealed that they were essentially identical indicating thatthe peak contained a single polysaccharide. Polysaccharide purified fromautoclaving extract gave similar molar ratios for rhamnose, galactoseand glucose (data not shown).

Comparative studies with a coaggregation-defective mutant. Acoaggregation-defective mutant, S. oralis PK1831, derived from S. oralisH1 was examined for the presence of a polysaccharide structurallyrelated to the CIP and an oligosaccharide similar or identical to thatisolated from wild type S. oralis H1. Cells of the mutant were grown andprocessed under conditions identical to those used with wild type cells.The properties of the mutanolysin released polysaccharide material fromthe coaggregation defective mutant of S. oralis H1 were very differentfrom CIP, i.e., this material failed to bind to the anion exchangecolumn, it failed to block coaggregation, and treatment with HF did notyield the coaggregation-inhibiting oligosaccharide similar to thatisolated from wild type cells (FIG. 5). This finding supports thefinding according to the instant invention, i.e., that thehexasaccharide is the determining factor in the coaggregation inhibitingactivity, or, in other words, that the hexasaccharide repeating unitderived from the CIP purified from the wild type Streptococcus oralis H1cell functions as the receptor for the adhesin from Capnocytophagaochracea ATCC 33596.

Quantitative inhibition assays. Quantitative inhibition assays (FIG. 6)were performed with mutanolysin extract, polysaccharide purified by ionexchange (from pooled Peak 2, FIG. 1) and HPLC purified oligosaccharide(see FIG. 2). These moieties produced a 50% inhibition of coaggregationat concentrations of 5.10, 1.79 and 0.44 mg/ml respectively. The freeoligosaccharide was therefore the most efficient inhibitor ofCapnocytophaga ochracea ATCC 33596- Streotococcus oralis H1coaggregation, as it was 4 times more effective than themutanolysin-released material. Oxidation of the purified polysaccharidewith periodate destroyed all coaggregation-inhibiting activity.

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What is claimed is:
 1. A method of inhibiting the build up of humandental plaque deposits in the human oral cavity by administering acomposition comprising the hexasaccharide of the formula I ##STR2##wherein "rha" designates rhamnose, "gal" designates galactose, and "glc"designates glucose, to the human oral cavity.
 2. A composition forinhibiting the build of human dental plaque deposits in the human oralcavity comprising the hexasaccharide of the formula I ##STR3## wherein"rha" designates rhamnose, "gal" designates galactose, and "glc"designates glucose, in admixture with a conventional carrier or diluentfor oral hygiene products.
 3. A composition according to claim 2 in theform of a tooth paste or mouth wash.
 4. A composition according to claim2 wherein the hexasaccharide is present in an amount of from 0.001 to 5%by weight.
 5. A method of inhibiting coaggregation between Streptococcusoralis ATCC 55229 and Capnocytophaga ochracea ATCC 33596 wherein thehexasaccharide of the formula I ##STR4## wherein "rha" designatesrhamnose, "gal" designates galactose, and "glc" designates glucose, ispreincubated with the Capnocytophaga ochracea.
 6. A method forinhibiting the adhesin from the gram-negative bacteria Capnocytophagaochracea ATCC 33596 wherein the hexasaccharide of the formula I ##STR5##wherein "rha" designates rhamnose, "gal" designates galactose, and "glc"designates glucose, is incubated with the bacteria.